Epithelial to mesenchymal transition influences fibroblast phenotype in colorectal cancer by altering miR‐200 levels in extracellular vesicles

Abstract Colorectal cancer (CRC) with a mesenchymal gene expression signature has the greatest propensity for distant metastasis and is characterised by the accumulation of cancer‐associated fibroblasts in the stroma. We investigated whether the epithelial to mesenchymal transition status of CRC cells influences fibroblast phenotype, with a focus on the transfer of extracellular vesicles (EVs), as a controlled means of cell–cell communication. Epithelial CRC EVs suppressed TGF‐β‐driven myofibroblast differentiation, whereas mesenchymal CRC EVs did not. This was driven by miR‐200 (miR‐200a/b/c, ‐141), which was enriched in epithelial CRC EVs and transferred to recipient fibroblasts. Ectopic miR‐200 expression or ZEB1 knockdown, in fibroblasts, similarly suppressed myofibroblast differentiation. Supporting these findings, there was a strong negative correlation between miR‐200 and myofibroblastic markers in a cohort of CRC patients in the TCGA dataset. This was replicated in mice, by co‐injecting epithelial or mesenchymal CRC cells with fibroblasts and analysing stromal markers of myofibroblastic phenotype. Fibroblasts from epithelial tumours contained more miR‐200 and expressed less ACTA2 and FN1 than those from mesenchymal tumours. As such, these data provide a new mechanism for the development of fibroblast heterogeneity in CRC, through EV‐mediated transfer of miRNAs, and provide an explanation as to why CRC tumours with greater metastatic potential are CAF rich.

for TGF-β content by exposure to MLEC cells. Luciferase activity was normalised to untreated (Ctrl) cells, which were assigned the value 1. Statistical significance was determined by two-tailed unpaired t-test.  conditioned with EVs from CRC cells at increasing concentrations (0.5, 1.5 and 4.5 x10 9 particles/ml) for 24h. EV-depleted medium was used as a control. (D) Conditioning of MRC5 cells with EVs from SW480 control and SW480-ZKD cells (0.5, 1, 1.5 and 4.5 x10 9 particles/ml) for 24h. HSP90 was used as an equal loading control. Representative of three experiments. For (C) and (D), band intensities for p-ERK1/2 and p-Akt473 were normalised to HSP90 and then to total ERK1/2 or total Akt, with the first band assigned the value 1. Values plotted are from three independent experiments.
Statistical significance (each band compared to the first band) was determined by two-tailed unpaired t-test (ns -not significant; * p<0.05; ** p<0.01; *** p<0.001).   fibroblasts were conditioned with DiD-labelled (far red, FL4 channel) SW480 (mesenchymal) or DLD1 (epithelial) EVs as described previously for three days and treated with TGF-b for an additional two days to initiate myofibroblastic differentiation. Fibroblasts were then collected, fixed, permeabilised and assayed for a-SMA immunoexpression. From left to right: total a-SMA, FL4 gating to identify cells with low and high EV uptake, a-SMA in EV low and EV high population. Mean fluorescence intensity (MFI) is indicated in each plot/ histogram. Figure S7. 3'UTR luciferase reporter assays for miR-200/ZEB1 targeting in MRC5 fibroblasts. Cells were transfected with ZEB1 3'UTR, mutant 3'UTR or control 3'UTR, together with miR200b/c or scrambled control. Luciferase activity was normalised to scrambled control, which was assigned the value 1. Statistical significance was determined by two-tailed unpaired t-test (ns -not significant; *   quantitatively using a gridded screen with Optical Density (OD) calculator. Values plotted are the means of nine (100 µm 2 ) fields of view from three independent sections. Statistical significance was determined by two-tailed unpaired t-test (ns -not significant; ** p<0.01; *** p<0.001) for A-C.

Cell lines
DLD1, HCT116, SW620 and SW480 human colorectal adenocarcinoma cells were procured from American Type Culture Collection. MRC5 human foetal lung fibroblasts were procured from The European Collection of Authenticated Cell Cultures. Hepa1-6 cells (metastatic mouse hepatoma) were purchased from German Collection of Microorganisms and Cell Cultures. All commercially acquired cell lines were characterised by STR profiling (Eurofins Genomics, Luxembourg) every six months.

MLEC assay
5x10 5 MLEC cells were seeded into each well of a 24-well plate. The following day, cells were treated with 0, 250, 500, 1000 or 2000 pg/ml TGF-β (cat no. 240-B; R&D Systems) to generate a dose-response curve. In parallel, cells were treated with conditioned medium (diluted 1:1 with fresh medium) from SW480 control or SW480-ZKD cells. All standards and experimental conditions were conducted in triplicate. At 24h, medium was aspirated and replaced with 100 μl Passive Lysis Buffer (cat no. E1941; Promega). Cells were then incubated at room temperature for 20 min on a rocker. 20 μl of each lysate was then transferred to each well of a 96-well plate, and 100 μl Luciferase Assay Reagent (cat no. E1500; Promega) was added to each well. Firefly luciferase activity was then measured using a luminometric plate reader (Varioskan LUX Multimode Microplate Reader; ThermoFisher). In parallel, 5 μl of cell lysate was used to measure protein concentration. Firefly luciferase activity was normalised by protein concentration for each well.
Membranes were then washed and incubated with horseradish peroxidase-conjugated secondary antibodies (polyclonal swine anti-rabbit (cat no. P021702-2) and polyclonal rabbit anti-mouse (cat no. P016102-2); 1:3000; Dako). Membranes were then washed again, and specific signal was visualised using SuperSignal West Dura (cat no. 34075; ThermoFisher) or West Femto (cat no. 34094; ThermoFisher) chemiluminescent detection kits. Where necessary, band intensity was quantified in Image J. Values shown are relative to b-actin/ HSP90 and normalised to the first lane of the blot, which was given the value 1. For phospho-ERK and phospho-Akt, values were normalised first to b-actin/ HSP90 and then to total ERK and total Akt respectively.

Intracellular staining for flow cytometry
Trypsinised cells were pelleted at 400g for 5 min, washed once with PBS, re-suspended in 4% paraformaldehyde and incubated for 20 min at room temperature. Next, cells were washed with PBS and resuspended in permeabilisation buffer (0.1% Triton-X100 in PBS) for 5 min. After another wash with PBS, cells were incubated in 0.5 ml blocking buffer (2% BSA in PBS). Primary antibody (α-SMA; 1A4; Sigma) was added to the blocking buffer at a dilution of 1:500. After a further incubation of 1h at room temperature, cells were washed twice with PBS, resuspended in blocking buffer containing 1:500 diluted fluorescent secondary antibody (Alexa 488-conjugated anti-mouse; cat no. A11011; Life Technologies) and incubated for 1h in the dark. Following another two washes with PBS, cells were analysed using FACS-Calibur (BD Biosciences).
A carbon coated formvar copper grid (cat no. TG50Cu; EM Resolutions) was placed on the droplet to immerse its coated side, and incubated for 30s at room temperature. Excess sample was blotted away using absorbent paper. The grid was then incubated with 10 μl negative stain (5% ammonium molybdate/ 1% trehalose) for 10s. Excess negative stain was removed by blotting. The grid was visualised at increasing magnification up to 135 000x using the Tecnai 12 microscope (FEI).

Nanoparticle Tracking Analysis (NTA)
The size distribution of EVs was measured by NTA (NS300; NanoSight), equipped with an EMCCD camera and a 405nm diode laser. Silica beads (100 nm diameter; cat no. 140120-10; Microspheres-Nanospheres) were used to calibrate the instrument. EV samples were diluted 1:5000 in PBS to optimise the particle number in the field of view. For each sample, five videos, each of 90s duration, were captured. Analysis was performed using the instrument software (NTA 2.3.0.15).

RNA extraction
Cellular RNA was isolated using the miRNeasy mini kit (cat no. 217004, Qiagen) and EV RNA using the miRNeasy micro kit (cat no. 217084; Qiagen), as per the manufacturer instructions. RNA quality and concentration was determined using the NanoDrop 8000 spectrophotometer (ThermoFisher).

MiRNA array
The Cancer MicroRNA qPCR Array with QuantiMir™ (cat no. RA610A-1; SBI System Biosciences) was used to profile CRC cells and EVs for 95 cancer-related miRNAs, as per manufacturer instructions.
Samples were processed in triplicate. A global mean normalisation method was used, as previously described (D'Haene et al. 2012). Combined mean 2 (-ΔΔCT) values for epithelial cells or EVs (DLD-1, HCT116 and SW620) were compared with mesenchymal (SW480) cells or EVs for each miRNA, to generate fold changes. MiRNAs for which 2 (-ΔΔCT) values were less than 0.1 in all samples were excluded. EV miRNA profiling data was deposited in Exocarta (Mathivanan et al. 2012).

RT-qPCR for miR-200
Total RNA was converted into cDNA using the Taqman Advanced miRNA cDNA Synthesis Kit (cat no. A28007; ThermoFisher). Input material was 4ng of total RNA (2 µl of 2 ng/μl). Expression levels were normalised to miR-423-5p (endogenous reference gene; based on literature (Link, Krohn, and Schumann 2019;Bignotti et al. 2016) and our own observations (Figure 3)), calculated from the triplicate of CT values, using the ΔΔCT method, and expressed relative to one of the samples that was assigned the value 1. Mean relative levels were calculated for each sample. Assay reference numbers were as follows: miR-200a-3p (478490_mir), miR-200b-3p (477963_mir), miR-200c-3p (478351_mir) and miR-141-3p (478501_mir). Mature miRNA sequences are shown in Table S1. For PCR reaction, the miR-Amp product was diluted 1:10 with RNase-free water, and 5 µl added to one well of a 96-well plate. This was combined with 10µL TaqMan® Fast Advanced Master Mix (2X), 1 µl TaqMan® Advanced miRNA Assay (20X) and 4 µl RNase-free water (total reaction volume 20 µl). Reactions were set up in triplicate with the following cycling conditions: 95 o C for 20s, followed by 40 cycles of 95 o C for 3s/ 60 o C for 30s, using the Applied Biosystems 7500 qPCR instrument. Table S1. Taqman Advanced miR-200 qPCR assays (Applied Biosystems/ ThermoFisher)

MiRNA, antagomiR and siRNA transfection of fibroblasts
MRC5 or NCFs (4 x 10 5 cells) were seeded into 6-well plates (day 0 staining was done by a histopathologist. Images were divided into 100 µm 2 squares (gridded screen) and the central nine squares of each image were analysed for positively and negatively stained CRC cells and fibroblasts. Optical Density (OD) calculator was used as a digital pathology platform. Signal localisation (nucleus-cytoplasm) was considered when identifying positive staining.

Dissociation of tumours into single cells:
The remaining tumours (nine from each group) were pooled together and dissociated into single cells for flow-sorting. Tumours were collected in ice-cold DMEM/F12 medium (cat no. 21331020; Gibco/ ThermoFisher) and then sterilised by immersion in 70% ethanol for 5s, followed by washing in two changes of regular DMEM. Tumours were then cut into fragments of 1 mm 3 and incubated at 37 o C for 2h with the digestion solution (0.125% collagenase (cat no. 10103586001; Roche); 0.0125% hyaluronidase (cat no. H3506; Sigma); 1 mM L-glutamine; 5% FBS; in 10ml DMEM/F12). The tissue was agitated every 30 min by vortexing for 1s. After 2h, the tissue was vortexed for 5s and then centrifuged at 350g for 5 min. The pellet was resuspended in 30 ml of HBSS (containing 2% FBS) and centrifuged at 350g for 5 min. This was repeated with HBSS without serum. The pellet was then resuspended in 5ml pre-warmed 0.25% Trypsin-EDTA (cat no. T4049; Sigma) and continuously mixed for 2 min by pipetting, to mechanically dissociate the organoid-like structures. 30ml of HBSS (+ 2% FBS) was added and the cell suspension centrifuged at 350g for 5 min.
2 ml of 5 mg/ml pre-warmed Dispase II (cat no. 4942078001; Roche), containing 20 μl of 10 mg/ml DNase I (cat no. 10104159001; Roche) was then added to the cell pellet. The cells were then pipetted up and down for 2 min to dissociate the remaining clumps. 30 ml of cold HBSS (+2% FBS) was then added and the cells centrifuged at 350g for 5 min. The pellet was resuspended in HBSS (+2% FBS) and filtered through a 40 μm cell strainer into a new tube. This was then centrifuged again at 350g for 5 min